TY - JOUR T1 - NT-mini, a recombinant tool for the study of Neurotrypsin functionality JF - bioRxiv DO - 10.1101/2020.10.01.322396 SP - 2020.10.01.322396 AU - Anselmo Canciani AU - Cristina Capitanio AU - Serena Stanga AU - Silvia Faravelli AU - Pascal Kienlen-Campard AU - Federico Forneris Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/10/02/2020.10.01.322396.abstract N2 - Neurotrypsin (NT) is a highly specific nervous system multi-domain serine-protease best known for its selective processing of the potent synaptic organiser agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. Something which, based on the available literature, likely stems from NT-based regulation of agrin signalling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, owing to the scarce molecular detail regarding NT activity and NT-agrin interactions. The difficult recombinant production of NT in its catalytically competent form is at the base of these limitations, and is currently constraining a more detailed molecular, biochemical and structural characterisation of the NT-agrin system. We have developed a novel strategy to reliably produce and purify a truncated but catalytically competent variant of NT called NT-mini. The characterisation of our construct highlighted almost wild type-like behaviour with comparable specificity, and conservation of modulation by calcium and heparin despite the lack of several accessory domains. With the data obtained from NT-mini it was then possible to identify NT’s heparin-binding domain, and discover a novel putative Zinc-based modulation of NT. Additionally, NT-mini allowed us to investigate the effect of NT activity on myotube formation in controlled cell-based experiments, evidencing a negative impact on myoblast fusion dependant on enzymatic activity. Collectively, this shows the viability of NT-mini as a model to study NT functionality, allowing to expand both in-vitro and “in-cellulo” investigations and providing a foundation to unravel the molecular underpinnings and biological significance of NT-agrin interactions.Competing Interest StatementThe authors have declared no competing interest. ER -